Size Exclusion Chromatography of Biopharmaceticals: Truth ... · Size Exclusion Chromatography of...

Size Exclusion Chromatography of

Biopharmaceticals:

Truth or Fiction

Christina Vessely

Senior Consultant, Biologics Consulting

Introduction

Aggregates have been of concern to regulatory agencies for a

number of years Linked to adverse events in patients

Injection site reactions

Anaphylaxis

Immunogenicity

Consequences

Discomfort

Permanent Damage

Death

Size Exclusion Chromatography has long been considered a

workhorse of the industry for the detection and quantitation of

aggregates Included on the vast majority of analytical release testing panels for

biotherapeutic products

Therapeutic Proteins

Antibodies

Peptides

Other

What is the value of SEC-HPLC?

Quantitative evaluation of species based on molecular weight Monomers

Dimers

High molecular weight species (HMW)

Relatively High Throughput Run times on the order of 20 – 40 minutes/sample

Not labor intensive Dilute and shoot

Initial prep of samples

Occasional monitoring of run

Data analysis

Integration parameters can be pre-programmed

Clean up

Validatability A well developed method will demonstrate

Relative Accuracy/Linearity

Precision (repeatability and intermediate precision)

Specificity

Orthogonal methods

So why is everyone at this meeting

talking about orthogonal methods?

At Best, SEC-HPLC tells only part of the story

with respect to aggregates and immunogenic

potential of a product

Deep down, none of us really trust the data we

get from our SEC-HPLC assays.

What are we missing?

Very large aggregates / Particles Larger aggregates may never enter the HPLC column

Filtered by the inlet frit of the column

Confirmation of molecular weight of each species QC laboratories often use a single detector

Molecular weight assignments are made based on

Proximity to the monomer peak

Assume the species to the left of the main peak is a monomer

Comparison to molecular weight standards

Assumes all species are similar in conformation

Natively unstructured protein

Globular protein

SEC Case Study – Pegylated Protein

• Comparison of two different samples

Monomer?

Fragment?

Aggregate?

SEC Case Study – Pegylated Protein

• Comparison of two different samples

Monopegylated

Unpegylated

Di-pegylated

Dimer

Why don’t we believe our results?

SEC-HPLC Parameters Column

Mobile Phase

Sample

Instrument

Environment



Porous solid particles

Separation is achieved by the amount of interaction / exclusion a

particular species has with the pores

Larger species will more likely be excluded from pores

Potential for surface interactions that may increase the tendency

to aggregate during elution

Column temperature can impact elution profiles

Assay is often run at “ambient” conditions

Ambient temperature can change depending on season, location

in laboratory, time of day

Columns degrade over time

May lose the ability to see certain aggregate species

Resolution may decrease, making accurate quantitation more difficult

Mobile Phase

Sample

Environment

Aggregate characteristics



Mobile Phase

Aqueous buffer, salt to reduce non-specific interactions

Colloidal Stability

pH

Aggregation is usually more prevalent close to isoelectric point

of protein

Less charge-charge repulsion

Salts

Increases in salt concentration can also decrease charge-

charge repulsion

Other additives

Organic modifier

Can increase or decrease prevalence of aggregates

Sample

Environment

Aggregate Characteristics



Mobile Phase

Sample

Is the sample in the HPLC vial the same as in the drug substance or

drug product container?

Dilution prior to injection

Concentration dependence of reversible aggregates

What is your diluent?

Environment




Mobile Phase

Sample

Environment

Temperature can influence elution profiles in SEC-HPLC

Many SEC-HPLC methods are run at “ambient” conditions

Ambient temperature can change depending on

Season

Location in the lab

Time of day




Mobile Phase

Sample

Environment


All aggregates are NOT created equal

Aggregates tend to be stickier than monomers

Potential that larger aggregates will be permanently adsorbed to

the solid phase of the column

Results in an underestimation of percent impurity of a sample

SEC-HPLC Method Development

Choose a column that is appropriate for your product Size range for column should match expected ranges for monomers,

dimers, and higher order molecular species

Evaluate multiple columns to determine the ability to resolve aggregate

species in your product

Confirm recovery of your protein from the column Different methods to accomplish this

Calculation based on area under the curve, absorbance, extinction

coefficient

If there are no interfering species in the mobile phase, may be simpler

to inject the protein in the presence and absence of column

Compare total peak area

Mobile phase compatibility Data from formulation development studies can be leveraged to improve

your SEC method

Impact of salt on aggregates

What is the mobile phase pH vs. isoelectric point?

What is the mobile phase pH vs. the formulation pH?


Performed forced aggregation studies Generate a “stable” aggregate

Agitation, with or without thermal stress

If you have surfactant in your formulation, you may have difficulty

generating aggregate

Freeze/thaw cycling

Prepare samples of different aggregation concentration (based on

measured concentration in stock)

Analyze in your SEC method

Confirm that you have linearity across a concentration range


Build consistency into your method Perform method robustness studies early to evaluate

Impact of slight but deliberate changes to mobile phase composition

Salt concentration

pH

Condition new columns before use

Most columns will have some level of non-specific interaction

Block non-specific binding

Evaluate column life and understand the signs of column degradation

Don’t count on ambient temperature to be consistent

Set column temp at 30C

Set smart system suitability criteria

Indicative of issues with column, instrument, or laboratory error

Should not be so restrictive that you are failing a “good” assay

Utilize reference standards

System suitability criteria should include an evaluation of reference

standards

Do they match typical profiles

Are there any unexpected peaks or out of trend results?


Use Orthogonal methods Do the results of your SEC-HPLC agree with other results?

Relevant orthogonal methods from your release panel

Appearance

SDS-PAGE

Denatures and dissociate non-covalent aggregates

Addition of reducing agent to dissociate covalent aggregates

If you are seeing aggregates by SDS-PAGE and not by SEC-

HPLC, you need to investigate

Particle methods

Again, particles may be filtered at the column inlet and therefore

would not be detected by SEC-HPLC

Don’t wait until late stage to apply extended characterization methods!!!!

SEC-MALLS

Use three detectors; UV, Refractive Index, and Multiangle light

scattering

Allows for specific determination of molecular weight of

aggregate species

Increased signal in MALLS detector for higher order aggregates

Confirmation of results

Cross verification studies (Analytical Ultracentrifugation) Forced aggregate study

Various concentrations of aggregate/monomer across range

Ideally range could cover at least 0.5 – 15% aggregate

Prepare samples and analyze in parallel

Samples should be run on the SEC method the same day as on AUC

Prevents the observations of different aggregate levels between the

two techniques resulting from different ages of samples

Samples should be run in the same laboratory if possible

Samples may be subject to agitation induced aggregation with

shipment to a contract laboratory

Do not be surprised if the methods don’t match!!!

It is likely to see higher levels of aggregate by AUC than by SEC

More critical to understand the relationship between the two methods

Evaluate slopes and trends with respect to aggregate levels and

types

What if my method is bad?

Define bad

Poor separation?

Not seeing aggregates that are observed in other assays?

No correlation between SEC and other orthogonal methods?

Further optimization

Are there other column types/chemistries to evaluate?

Changes to mobile phase

Addition of other additives

Admit defeat

What other methods can I consider?

Alternatives

Asymmetrical flow field flow fractionation (AF4) Asymmetrical flow field flow fractionation (AF4)

Separation is achieved in the absence of a column

Uses flow in two directions (parallel to channel and perpendicular to

channel) to achieve separation

First step is focusing

Increased concentration of protein into a small band – can promote

aggregate formation for some products

AUC????? May be necessary in some cases

Low throughput

Higher limit of quantitation

Typically require >1% aggregate before it can be reliably detected

Formulation excipients can increase the quantitation limit

Example, sucrose alters viscosity of formulation and therefore

impacts rate of sedimentation

Limit of quantitation is closer to 3% for formulations with high

levels of sugars

Conclusions

A good SEC-HPLC method is a critical part of the analytical toolbox

for biotherapeutics Invest in method development at early stages of the program

Critically evaluate the quality of the data

SEC-HPLC should be used in conjunction with orthogonal

techniques Complementary techniques to give a more complete picture of aggregate

profiles of a solution

Apply critical evaluation of results to ensure the assays are telling a consistent

story

If your data from orthogonal techniques are not in agreement, you need to

investigate

Issue with one method or the other?

Cross-verification to understand / establish relationship between

results

Questions???

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